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. 2022 Feb 9;13:762. doi: 10.1038/s41467-022-28454-x

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — A U2OS-Sec61β cells incubated with DiD-labelled Lp-EVs. Live cell image shows co-localization of Lp-EVs with ER and mitochondria (left and magnifications) and with acidic organelles (right and magnifications). Representative images of n = 3. White: DiD-labelled Lp-EVs; Green: ER; Blue: Nucleus; Red: Mitochondria; Orange: acidic organelles. B Quantification of co-localization events over time. Live cell image acquisition of multiple fields per well of infected U2OS-Sec61β cells was performed on an automated confocal microscope and the mean fluorescence intensity (MFI) of GFP, Mitotracker and Lysotracker, and Lp-EVs was analyzed, suggesting that they co-localize with ER and lysosomes. Data are presented as (mean) SD with each dot representing the mean of up to 600 independently analyzed cells. For statistical analysis an unpaired t-test was performed (two-tailed; p < 0.05 significant). Source data provided as Source data file. C U2OS cells labelled with Rab5 and Rab7 CellLight incubated with DiD-labelled Lp-EVs. Co-localization of Lp-EVs with Rab5⁺ early endosomes (EE) 4.5 h post-infection and with Rab7⁺ late endosomes (LE) 5 h post-infection. Live cell image acquisition of multiple fields per well of infected U2OS cells was performed over the time at 37 °C (5% CO₂) on an automated confocal microscope. Representative images of five independent experiments. White: DiD-labelled Lp-EVs; Blue: Nucleus; Red: Rab5; Green: Rab7. D Quantification of Lp-EVs co-localizing with EE and LE were determined during 5 h of infection. Data are presented as (mean) SD with each dot representing the mean of around 500 independently analyzed cells. Source data provided as Source data file. E Quantification of purified HiBiT-GroEL EV content delivery within LgBiT-THP-1 acceptor cells, +/− Bafilomycin A1 (BA1) or Cytochalasin D (CD) treatment. Negative control, LgBiT-THP-1 cells incubated with purified Lp-EVs (no HiBiT). The luminescence in the negative control was defined as 0%. Bafilomycin A1 (loss of endosomal acidification) and Cytochalasin D (inhibition of actin polymerization) significantly reduces the content delivery into the host cell. Error bars represent the (mean) SD of at least n = 8 biological repeats. For statistical analysis, a two-way ANOVA analysis was performed with p < 0.0001 (****). Source data provided as Source data file.